Phosphate-dependent glutaminase is activated and dimerized by various anions including phosphate. Experiments in which the glutaminase was covalently attached to Sepharose as either monomers or dimers, established that dimerization is essential for catalytic activity. The stoichiometry and kinetics of inactivation of the glutaminase with either of two affinity labels, 6-diazo-5-oxo-l-norleucine and L-2-amino-4-oxo-5-chloropentanoic acid, has been characterized. The former acts as an analog of glutamine and binds only to the active dimer, whereas the latter inhibitor acts as an analog of glutamate and preferentially reacts with the inactive monomeric form of the enzyme. We are currently characterizing the glutamate inhibition of this enzyme. The phosphate-dependent glutaminase is associated with the inner mitochondrial membrane. In order to determine whether transport or enzyme activity is the rate limiting step in glutamine metabolism, we have developed methodology to selectively inhibit the glutaminase in intact mitochondria using either of the two affinity labels. This has made it possible to now measure transport independent of metabolism. In addition we have developed methodology to use F(ab)2 antibodies to selectively precipitate the glutaminase from detergent solubilized mitochondria. We have recently established that the purified glutaminase consists of a series of related peptide fragments (Mr equals 55-70,000) which are produced by partial proteolysis of the native enzyme (Mr equals 81,000) during its solubilization and purification. We are now developing procedures to purify the undegraded enzyme. In addition we plan to use immunoprecipitation procedures to study the turnover of the glutaminase in normal and acidotic states.